Tissue culture and rapid propagation technology for Gentiana rhodantha

Abstract Gentiana rhodantha is a perennial herb of the genus Gentiana (Tourn.) L. This study was novel in establishing a regeneration system of G. rhodantha using young leaves as explants on the Murashige and Skoog (MS) medium supplemented with different plant growth regulators (PGRs). The roots, stems, and leaves of G. rhodantha were used as explants. The effects of the optimal explant disinfection method, type of explant used, concentrations of PGRs added to the culture media on tissue culture, and rapid propagation of G. rhodantha were studied. The results showed that the optimal disinfection method for stems and roots consisted of disinfection using 75% ethanol for 50 s, followed by 4% sodium hypochlorite (NaClO) for 10 min. The optimal disinfection technique for leaves consisted of disinfection using 75% ethanol for 50 s, followed by 4% NaClO for 8 min. Root explant was the most suitable for inducing the callus of G. rhodantha on the MS medium supplemented with different PGRs. The optimal conditions for callus induction included 1.0 mg/L 6-benzylaminopurine (6-BA) and 0.5 mg/L α-naphthalene acetic acid (NAA). The callus induction rate using the root explant reached 94.28%. MS supplemented with 2.0 mg/L 6-BA and 0.1 mg/L NAA was the optimal medium for inducing adventitious shoots from the callus of G. rhodantha. The best medium for propagation and plantlets strengthening was MS supplemented with 0.8 mg/L 6-BA and 0.3 mg/L NAA, and the propagation index was 8.62. MS supplemented with 0.3 mg/L 3-indolebutyric acid was the best culture medium for inducing the rooting of adventitious buds, with the maximum rooting rate reaching up to 100%.


Introduction
Gentiana rhodantha Franch. is a perennial herb of the genus Gentiana (Tourn.) L. and a newly catalogued medicinal herb in the 2015 Chinese Pharmacopoeia [1]. G. rhodantha is an endemic plant in China and rich in compounds such as mangiferin, flavonoids, and sterols. Mangiferin is known for its definite efficacy in relieving cough and inflammation. In recent years, the demand for G. rhodantha as a raw material has grown. It is part of the folk tradition to use G. rhodantha to treat pneumonia, bronchitis, and hepatitis [2,3]. G. rhodantha plantlets are short and have many branches. This plant blooms in winter, and the flowers are bright-colored and last long. It can also be used for urban landscaping in winter or grown in pots for indoor decoration [4]. However, seed germination can be tricky in the case of G. rhodantha. Given its reproductive capacity, G. rhodantha is commercially propagated by cutting its top buds. However, this propagation method is usually associated with slow rooting and poor growth. Sexual reproduction, which involves a long cycle, can be considerably restricted by the natural environment. Besides, the reproduction rate and the plant quality are low with split cultivation, making G. rhodantha unsuitable for mass production [5]. The conventional propagation techniques for G. rhodantha are time-consuming, involve complex procedures, and have low yields, and therefore can hardly meet the market demand. On the contrary, the propagation system of G. rhodantha is suitable for mass production to meet the increasing market demand [6].
The recent studies on G. rhodantha mainly focus on the following aspects: germplasm resource investigation [7], chemical composition analysis [8,9], pharmacological research [3][4][5][6][7][8][9][10][11][12], and localization of suitable areas [13]. On the contrary, few studies involve tissue culture of G. rhodantha. Zhong built a rapid propagation system of G. rhodantha using nodal segments [14]. The callus induction and adventitious bud differentiation for G. rhodantha are much less discussed. Tissue culture is featured by the high propagation index and controllability of culture conditions. These two advantages can greatly accelerate the propagation of G. rhodantha, contributing to the popularization of this species.
In this study, the young and tender roots, stems, and leaves of G. rhodantha were used as explants. The callus induction, adventitious shoots differentiation, and rooting of this plant species were examined using the optimal disinfection method and the optimal hormone ratio. We replaced the conventional ethanol-corrosive sublimate combination for disinfection with sodium hypochlorite (NaClO) to reduce environmental pollution. On this basis, we built the rapid propagation system of G. rhodantha, which laid the foundation for the rapid propagation of this species, alleviated the shortage of wild G. rhodantha resources, and created favorable conditions for developing ornamental G. rhodantha.

Experimental materials
The plants of G. rhodantha were brought from Daba Village, Huize County, Qujing City of Yunnan Province. They were transported to the Mianyang Normal University of Sichuan Province, People's Republic of China (104°78′E, 31°5′N). All culture media were supplemented with 30 g/L sucrose and 7.5 g/L agar. All media were adjusted to pH 5.8 with 0.1 N sodium hydroxide solution before autoclaving at 121°C for 20 min. All explants and plantlets were cultured at 25 ± 2°C under 16 h photoperiod and 2,000 lx light intensity.

Experimental method 2.2.1 Explant disinfection
The roots, young stems, and young leaves of G. rhodantha were used as explants. The explants were washed with running water for 1-2 h and then rinsed with sterile distilled water for 3 min.
On an ultraclean workbench, the stem, root, and leaf explants were decontaminated under aseptic conditions using 75% ethanol for 40, 50, and 60 s, respectively. Then, the explants were dipped in 4% NaClO solution and shaken for 8, 10, and 12 min, respectively. Next, the explants were washed using sterilized water five to six times and cut into 1 cm-long segments. They were inoculated into the Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 6-benzylaminopurine (6-BA) and 0.5 mg/L α-naphthalene acetic acid (NAA). The orthogonal experimental design was adopted, with nine different treatments established. One explant was inoculated into each bottle. Ten explants were inoculated for each treatment, thus totaling 90. After 1 month, contaminated explants for each treatment were identified and their optimal disinfection duration was determined. The optimal disinfection was used in the subsequent experiments.

Screening for the optimal culture medium for callus induction
The MS medium was supplemented with different concentrations of 6-BA (at the concentration of 0.5, 1.0, and 1.5 mg/L, respectively) and NAA (at the concentration of 0.5, 1.0, and 1.5 mg/L, respectively) for the stem, root, and leaf explants. Nine treatments were set up using the orthogonal design. After disinfection, three sterile explants were inoculated into one culture bottle. Each treatment encompassed three bottles, with three replicates. Thus, 81 bottles were used in total. The growth status was recorded on Day 30. The explants most suitable for callus induction and the optimal hormone concentration combination for this purpose were identified.

Screening for the optimal culture medium inducing adventitious shoot regeneration
The vigorously growing calluses were inoculated to the MS media supplemented with different concentrations of 6-BA (at a concentration of 1.5, 2.0, and 2.5 mg/L, respectively) and NAA (at a concentration of 0.1, 0.2, and 0.3 mg/L, respectively) for the stem, root, and leaf explants. Nine treatments were set up using the orthogonal design. The explants were inoculated into three bottles for each treatment (under the same culture conditions as earlier), with three replicates. Thus, 81 bottles were used in total. The growth of adventitious buds was observed, and the induction rate was estimated 3 weeks later. The optimal culture medium for inducing adventitious bud differentiation from the callus of G. rhodantha was identified.

Screening for the optimal culture medium for the proliferation of adventitious shoots
The vigorously growing adventitious shoots were inoculated into the MS media supplemented with different concentrations of 6-BA (at a concentration of 0.8 and 1.2 mg/L, respectively) and NAA (at a concentration of 0.1, 0.3, and 0.5 mg/L, respectively). Six combinations were set up using the orthogonal design. The explants were inoculated into three bottles for each treatment, with three replicates. Thus, 54 bottles were used in total. After 1 month, the growth status was observed, and the propagation index was calculated.

Screening for the optimal culture medium for rooting
The vigorously growing adventitious shoots were inoculated to the MS media supplemented with IBA at a concentration of 0.1, 0.3, and 0.5 mg/L, respectively. Thus, three treatments were set up. The shoots were inoculated to three bottles for each treatment (under the same culture conditions as earlier), with three replicates. Thus, 27 bottles were used in total. The root growth was observed for 20 days, and the growth status was evaluated and subjected to statistical analysis.

Data processing
The data were statistically processed using Excel 2007 and SPSS 17.0. The experimental results were subjected to analysis of variance. 3.2 Effects of plant growth regulators on the rapid propagation of G. rhodantha

Effects of plant growth regulators on callus induction from different explants of G. rhodantha
The leaves gradually thickened and turned brittle in the last week of tissue culture. A few lateral buds began germinating in the stem segments, and the cut in the stem node gradually bulged outward. The roots turned from light yellow to green. Browning occurred in most leaves on Day 12 of tissue culture. The green color receded in the surviving leaves, and the callus grew in the cut ( Figure 1a). Yellow, transparent calluses were seen at the cuts in the rhizome and some stem segments (Figure 1b and c). The lateral buds grew in the stem segments ( Figure 1d). Later, the callus gradually covered the entire rhizome ( Figure 1e). After 40 days of tissue culture, the rhizome was enveloped by the callus, as shown in Figure 1f. The callus of the stem node grew slowly (Figure 1g). After 1 month, the calluses were compared between different treatments and explants. The performance of callus induction varied significantly across different combinations of hormone concentrations. Among the three types of explants, the number of browning leaves was far higher than that of browning calluses from stem nodes and roots. However, the growth rate of leaf calluses was far lower than that of calluses from stem nodes and roots. The calluses induced from different explants varied in color, density, and texture. The calluses induced from leaves were mostly yellowish white, with a hard, compact texture. The calluses induced by the stem and root segments were mostly yellowish green, with a loose texture and fast growth. As shown by the statistics in Table 2, the number of browning leaves was far higher than the number of browning calluses induced from roots and stem nodes. Therefore, yellowish-green calluses with a loose structure and fast growth could be induced by inoculating the explants to media containing 1 mg/L 6-BA and 0.5 mg/L NAA. Such calluses could be induced from roots and stem nodes with high efficiency, with the induction rate reaching 94.28%.

Effects of plant hormone concentration on
adventitious shoot regenerations from the callus of G. rhodantha The vigorously growing calluses induced from stems and roots were inoculated into the culture media for inducing adventitious shoots. The calluses grew slowly initially, and the texture became more impact. The bud points appeared on the surface of calluses about 1 week later. The adventitious roots induced from the injured roots grew vigorously about 2 weeks later. However, no adventitious shoots were induced (Figure 2a). Some calluses even died over time (Figure 2b). No adventitious shoots were formed after 40 days of culture; the roots thrived and then gradually died (Figure 2c). These observations might be related to the inappropriateness of the hormones used to induce callus differentiation from the roots. When calluses were induced from stems, the induction rate of adventitious shoots increased with the increase in the concentration (Table 3). However, the induction rate of adventitious shoots decreased as the concentration of 6-BA continued to increase beyond 2 mg/L. No adventitious shoots were induced from stem calluses in the media containing a high concentration of NAA and a low concentration of 6-BA. Besides, the adventitious shoots grew vigorously, with the induction rate of adventitious buds being 0. The adventitious shoots were vitrified when the concentration of Stem 60  8  20  11  10  11  17  Stem  60  10  20  12  8  13  18  Stem  60  12  20  10  7  15  19  Root  40  8  20  15  15  7  20  Root  40  10  20  13  13  9  21  Root  40  12  20  10  10  10  22  Root  50  8  20  10  9  7  23  Root  50  10  20  7  7  8  24  Root  50  12  20  8  6  11  25  Root  60  8  20  11  10  10  26  Root  60  10  20  12  8  12  27 Root 60 12 20 10 5 13  Notes: The data are expressed as mean ± standard deviation. The significance level is set to 0.05 (the same below).
Tissue culture and rapid propagation technology for Gentiana rhodantha  5    6-BA increased to 2.5 mg/L (Figure 2l). The induction of shoots from stem calluses was the best after treatment with 2.0 mg/L 6-BA and 0.1 mg/L NAA (Figure 2g). The difference was statistically significant compared with that from root and leaf calluses. The shoot regeneration rate reached 86.25%.

Effects of hormone concentration on the propagation of adventitious shoots from G. rhodantha
Adventitious shoot clusters induced from stems were cut into small pieces of equal sizes along with the corresponding calluses. They were inoculated into the multiplication-inducing culture medium. The propagation index and growth status of adventitious shoots were assessed after 1 month of tissue culture. On this basis, the culture medium most suitable for propagation and shoot strengthening was determined. As shown in Table 4

Effects of hormone concentration on the rooting of regenerated shoots
As shown in Table 5, all three concentrations of IBA could induce the rooting of regenerated shoots, and the rooting rate was consistently 100%. The roots became shorter and more slender under a high IBA concentration, and browning occurred with the increase in the IBA concentration. The average root length per treatment, and the growth and development morphology of roots were compared across the treatments. The MS medium supplemented with 0.3 mg/L IBA was found to be the most suitable for inducing the rooting of regenerated shoots of G. rhodantha, which resulted in an average root length of 2.53 cm ( Figure 4).

Discussion and conclusion
The presence of endophytes in Gentiana plants [15] usually results in a high explant contamination rate during tissue culture [16][17][18][19]. We determined the optimal disinfection method for the stem and root segments, which involved disinfection with 75% ethanol for 50 s, followed by 4% NaClO for 10 min. For leaves, the optimal disinfection method consisted of disinfection with 75% ethanol for 50 s, followed by 4% NaClO for 8 min. The stems, roots, and leaves were then used as sterile explants after the disinfection procedure. The present study used leaves, stems, and roots as explants. In the explants disinfection experiment, we found that the leaves, young roots, and young stems of G. rhodantha were more tender and susceptible to browning and death. The stem and roots had lower browning rates and higher survival rates than the leaves. This finding agreed with the results obtained by Huo [18] and Guo et al. [20]. Wang [19] found that the callus induction rate of young leaves in Conyza blinii H. Lev was higher than that of old leaves, although the differentiation rate remained low. This result agreed well with our finding of a lower induction rate and higher browning rate of the leaf calluses of G. rhodantha. This was probably because the Gentiana plants are rich in flavones, leading to a higher possibility of browning.
The type and concentration of exogenous hormones are closely related to the number and growth status of adventitious shoots induced from plants. The ratio of cytokinin to auxin is a key factor determining callus differentiation. In the present study, the differentiation rate of adventitious shoots from stems first increased and then decreased as the 6-BA concentration increased. As the 6-BA concentration increased to 2.5 mg/L, the adventitious shoots were shorter, slender, and severely vitrified. That is, an excessively high concentration of 6-BA had an inhibitory effect on adventitious shoot differentiation. No adventitious shoots were differentiated, and the root system grew exuberantly under the combination of low-concentration 6-BA and high-concentration NAA. The root-induced callus treated with the conventional hormone combination of 6-BA and NAA had no differentiation of adventitious shoots. No adventitious shoots germinated, and the roots grew vigorously under the joint action of a low concentration of 6-BA and a high concentration of NAA. The following conclusions were drawn from these observations: (1) G. rhodantha is a perennial herb with developed roots and remarkable rooting ability; therefore, many adventitious roots were induced from the root callus, but no adventitious buds were germinated [21].
(2) Root formation using high concentration of NAA could be due to the known effect of auxin, i.e., high auxin concentrations promote root formation. Thus, only a proper ratio of auxin and cytokinin can induce root and shoot regeneration.
During secondary culture, an increase in the propagation index of adventitious shoots depended on the  Notes: The data are expressed as mean ± standard deviation. The significance level is set to 0.05 (the same below).
increase in cytokinin concentration. However, as the cytokinin concentration continued to increase, the adventitious shoots showed poor growth and became shorter and slender, making the root culture difficult. Even if the roots were formed, the transplanted tissue culture plantlets were slender and had low stress resistance. Such plantlets might not survive and require plantlets-strengthening culture. According to our experiment, the MS supplemented with 0.8 mg/L 6-BA and 0.3 mg/L NAA culture medium was more suitable for propagation by adventitious shoots and plantlets strengthening, with the maximum propagation index being 8.6. Our experiments showed that G. rhodantha had a good rooting ability. The rooting rate was consistently 100%, regardless of the root-inducing medium used. Besides, rooting was also observed in the adventitious bud and propagation induction experiments. MS supplemented with 0.3 mg/L IBA was the optimal culture medium for inducing the rooting of adventitious buds from G. rhodantha, with a rooting rate of 100%. This was probably because Gentiana plants were perennial plants with strong rooting ability.
The growth hormones, explant type, and type of endogenous hormones in plants have a significant influence on the regeneration capacity of G. rhodantha. Our results showed that the adventitious shoots could be differentiated from the callus induced from stem explants, and rooting was successfully induced. The rapid propagation system of G. rhodantha was built on this basis, providing technical references for rapid growth of plantlets and large-scale cultivation.